Neoplastic transformation causes a selective decrease in the rate of fibronectin mRNA synthesis in fibroblast cells. Our objective is to increase our understanding of the molecular mechanisms that regulate human fibronectin gene expression in neoplasia. A human fibroblast cell line which is temperature sensitive for neoplastic transformation will be used in combination with molecular biological and genetic methods to A) identify the fibronectin gene DNA sequences responsible for decreased fibronectin mRNA synthesis in neoplastic transformation, B) identify factors acting on these sequences, and C) isolate the genes encoding these factors. The specific aims are: 1) To isolate the human fibronectin gene promoter by "sandwich" hybridization screening a human genomic DNA library with complexes of fibronectin mRNA and labeled, antisense fibronectin transcripts. Primer extension will locate the mRNA start site. 2) To prepare hybrid gene constructions in retroviral vectors so that the fibronectin gene promoter region controls the expression of an assayable "reporter" molecule. These constructions will be used to infect a human cell line which is temperature-sensitive for transformation and was developed for this purpose. Differential expression of the "reporter" molecule at the permissive and non-permissive temperatures will permit definition of the DNA sequences responsible for diminished fibronectin expression in neoplastic transformation. 3) To detect trans-acting factors which interact with fibronectin promoter DNA. A protein-DNA binding assay will be used to detect and purify proteins that bind specifically to these DNA sequences. A genetic approach will also be used to identify these factors. A retrovirus in which a selective marker is under the control of a defective fibronectin promoter regulatory region will be constructed. Temperature-sensitive cells infected with this virus will not exhibit differential expression of the marker at the permissive and non-permissive temperatures. Mutagenized cells will be selected to restore temperature-sensitive expression of the marker. 4) To isolate genes encoding the trans acting factor(s) by making antibodies against the purified proteins and using the antibodies to screen a lambda gt11 DNA expression library. The health relatedness of this project derives from its potential contribution to our understanding of the basic molecular mechanisms that regulate gene expression in neoplastic transformation.